In order to propagate our genome, our cells need to divide accurately over many generations; errors in the cell division process are linked to a wide variety of cancers. The fundamental structure of cell division is the self-organized assemblage of microtubules which adopts a bipolar configuration in metaphase and a central spindle upon entry into anaphase. This structure is subjected to numerous forces throughout mitosis, and must provide stability while remaining flexible and compliant to the highly motive environment. The key players involved are microtubules, motors, and non-motor microtubule-associated proteins (or MAPs), and many of their biochemical properties have been studied extensively. Much less is known about the role mechanical force plays in regulating the spindle's structural properties. This research project will utilize a single-beam optical trap in conjunction with two-color TIRF (total internal reflection fluorescence) microscopy in order to exert a force of known magnitude and direction on a microtubule structure that is cross-linked by PRC1 (a human non-motor MAP) and simultaneously visualize the response of this structure to the mechanically applied tension. It is known that PRC1 dimers selectively bind anti-parallel microtubules and localize predominantly at the central spindle midzone in anaphase. The question of how this cross-bridge responds to the forces present in vivo throughout cell division is still unanswered. Additionally, the role of specific protein domains and residues in contributing to organizational stability/flexibility is not fully known. The use of truncated and mutated constructs will help elucidate the mechanistic properties of the protein/microtubule unit. PRC1 is also known to recruit proteins, such as kinesins and kinases, to the spindle midzone. One such motor, kinesin-4, has been shown to work together with PRC1 as a minimal protein module to maintain a fixed midzone length. Kinesin-4 is a plus-end directed motor, which inhibits the growth of dynamic microtubules. The question of how this motor's recruitment and activity is modulated by the forces generated during cell division is unanswered. This protein module will be reconstituted in vitro, where force will be applied along the microtubules and the response of both proteins at the midzone will be measured in order to determine the role that force plays in regulating both the motor's activity and the length of the midzone overlap. In addition to the proposed research, a significant component of the fellowship period will entail a training program at Rockefeller University consisting of coursework, frequent seminars in biological and clinical research, and extensive instruction in biochemical, cell biology, and fluorescence imaging techniques in the research lab. Throughout the progression of this project, outstanding training in many biological and biophysical methodologies and techniques will be acquired, resulting in the attainment of a broad range of highly interdisciplinary skills by the conclusion of the fellowship period.